Summary Respiratory syncytial virus (RSV) is the primary cause of infant hospitalization from infectious diseases in the United States. Regular re-infection of adults can occur throughout life during seasonal epidemics, but can be life-threatening especially to the elderly and the immunocompromised. Despite extensive research, no vaccine protection is available and current antibody therapy-based immunoprophylaxis remains reserved for high-risk patients. Recognizing the unmet clinical need for efficacious, applicable, and well-tolerated RSV therapeutics, it is the goal of this project to pursue a rigorous preclinical characterization and de-risking program of two orally efficacious RSV polymerase inhibitor classes that we have identified in previous work and pilot studies. Having pioneered RSV reporter virus technology and completed large-scale high-throughput anti-RSV drug screening campaigns, we have identified two structurally and mechanistically distinct hit classes that both inhibit the RSV RNA-dependent RNA polymerase (RdRP) complex, a novel uridine ribonucleoside analog and a non-competitive inhibitor of initiation of RdRP-mediated RNA synthesis at the promoter. Current leads show potent activity against RSV reporter strains and clinical isolates, nanomolar inhibitory concentrations in disease-relevant well-differentiated primary human airway epithelia cultures, good pharmacokinetic (PK) profiles with sustained plasma concentrations, and good preliminary tolerability. Pilot studies have established proof-of-concept of oral efficacy in the mouse model of RSV infection, reducing lung viral load and hallmark clinical signs of RSV bronchiolitis. This project will pursue the RSV polymerase target in a two-pronged strategy, developing the substrate-analog and non-competitive inhibitor classes simultaneously to proactively mitigate the risk of early stage failure or lay the experimental foundation for future use as companion drugs. The initial approach will be tailored individually to either series, designed to identify and address potential class-specific liabilities early in the process. The ribonucleoside analog lead has shown sustained tissue concentrations of the active triphosphate form and sterilizing oral anti-RSV efficacy. In preparation of formal development, this class will be subjected to mechanism of action characterization, resistance profiling against the RSV target, and assessment of off-target effects (aim 1). The first-generation non-competitive inhibitor lead has been successfully resistance, cytotoxicity, and mechanism profiled, but must be subjected to final structure and QSAR-guided synthetic optimization of potency and PK properties to uncover its full antiviral potential (aim 2). Emerging confirmed leads of either class will be de-risked using the mouse model of RSV infection, pathogenesis of compound-experienced RSV populations will be assessed, and PK profiles correlated with performance in primary human airway epithelium cultures to inform simulations of the impact of physiological, dynamic drug concentrations on antiviral efficacy and safety margin in relevant human tissue models (aim 3).